JP6913454B2 - How to purify volatile organochlorine compounds - Google Patents

Description

The present invention relates to a method for purifying volatile organochlorine compounds. In particular, the present invention relates to a method of purifying volatile organochlorine compounds in the presence of hydrogen peroxide using bacteria of the genus Rhodococcus.

Purification techniques using microorganisms are widely used in activated sludge methods, anaerobic treatment methods, etc. in wastewater treatment. Further, in recent years, a technique (bioremediation) for purifying soil and groundwater contaminated with harmful chemical substances by microorganisms has been attracting attention as a method having a small environmental load and purification cost.

For example, purification techniques using anaerobic bacteria have been reported. Specifically, in order to purify groundwater contaminated with chlorinated ethylenes such as trichlorethylene and cis-1,2-dichloroethylene, organic substances that serve as nutrient sources for bacteria are injected into the aquifer from purification wells and the like. , A technique of making groundwater into an anaerobic environment, activating anaerobic dechlorinating bacteria, and purifying chlorinated ethylene into harmless ethylene has been widely put into practical use (Non-Patent Document 1). However, in this technique, dechlorinating bacteria replace chlorine in chlorinated ethylene one by one with hydrogen, so that vinyl chloride monomer is always produced as an intermediate product in the purification process (Fig. 1). Vinyl chloride monomer was added to the groundwater environmental standard item in 2009 by the Ministry of the Environment. Since the regulated concentration of vinyl chloride monomer is one order smaller than that of trichlorethylene and cis-1,2-dichloroethylene, vinyl chloride monomer that greatly exceeds the environmental standard value may be generated in groundwater depending on the progress of purification. , Secondary groundwater pollution may occur.

In addition to purification technology using anaerobic bacteria, purification technology using aerobic bacteria has also been reported. As aerobic bacteria, the existence of aerobic bacteria capable of decomposing trichlorethylene, cis-1,2-dichloroethylene and the like has been widely reported. The aerobic dechlorination reaction is mainly carried out by toluene-utilizing bacteria, phenol-utilizing bacteria, methane-utilizing bacteria and the like that grow using toluene, phenol, methane or the like as a single carbon source. The oxidase produced by these bacteria when using toluene, phenol, methane, etc. as a carbon source under aerobic conditions has a wide substrate specificity. For example, in the case of methane oxygenase, when trichlorethylene is present together with methane, the co-metabolization (cooxidation) reaction of trichlorethylene proceeds at the same time in addition to the oxidation of methane, and the dechlorination reaction of trichlorethylene is promoted. Are known. However, the carbon source (electron donor) required to promote these co-metabolism reactions is a harmful substance such as toluene or phenol, or a substance that is difficult to dissolve in water such as methane. The problem was that there was no effective means of supplying carbon sources to the underground environment.

A purification technique using bacteria that does not require a carbon source has also been reported (Patent Document 1). Patent Document 1 discloses a purification technique using a bacterium (JMP1000 strain) that does not require a carbon source and oxygen supplied from a hydrogen peroxide solution. The JMP1000 strain is disclosed to be resistant to 1,000 ppm (0.1%) hydrogen peroxide solution. Specifically, in Patent Document 1, a medium containing 1,000 ppm of hydrogen peroxide and ^{1 ml of a bacterial suspension of JMP1000 strain (cell concentration: 3.1 × 10 7} cfu / ml) were added to a vial. After completely sealing, trichloroethylene and trans-1,2-dichloroethylene were added with a gas tight syringe so that the concentration in the liquid was 10 ppm (10 mg / l), and the mixture was shake-cultured at 20 ° C. for 80 hours. It is disclosed that 59% decomposition was carried out and trans-1,2-dichloroethylene was decomposed by 71%. The resolution of the JMP1000 strain is not sufficient, and trichlorethylene and trans-1,2-dichloroethylene cannot be completely decomposed in about 4 days. Patent Document 1 does not disclose the decomposition of cis-1,2-dichloroethylene by the JMP1000 strain.

Japanese Unexamined Patent Publication No. 10-295366

Chemistry and Biology, Vol. 49, No. 4, pp. 256-260, 2011

As described above, many methods for decomposing volatile organic chlorine compounds such as chlorinated ethylene using microorganisms have been reported, but there is still room for improvement.

Therefore, an object of the present invention is to provide a new method for purifying (decomposing) a volatile organic chlorine compound.

As a result of diligent studies by the present inventors, by using a bacterium having a catalase-producing ability and an ability to decompose a volatile organochlorine compound in the presence of oxygen, and hydrogen peroxide, the bacterium acts as a catalase. It was found that volatile organochlorine compounds can be efficiently decomposed by using oxygen supplied from hydrogen peroxide.

That is, the present invention includes the following.
[1] A bacterium having a ability to generate catalase and an ability to decompose a volatile organochlorine compound in the presence of oxygen is allowed to act on the volatile organochlorine compound in the presence of hydrogen peroxide to cause the volatile organochlorine compound. A method for purifying volatile organochlorine compounds, which comprises a decomposition step of decomposing hydrogen peroxide.
[2] The method according to [1], wherein the volatile organochlorine compound is trichlorethylene and / or cis-1,2-dichloroethylene.
[3] The method according to [1] or [2], wherein the bacterium is a bacterium belonging to the genus Rhodococcus.
[4] The method according to [3], wherein the bacterium belonging to the genus Rhodococcus is a Rhodococcus jostii RHA1 strain.
[5] The method according to any one of [1] to [4], wherein the volatile organic chlorine compound is contained in the groundwater and the groundwater is purified in the original position.
[6] The method according to [5], wherein the hydrogen peroxide disappears and is converted into oxygen within 3 days after being introduced into the groundwater.
[7] The hydrogen peroxide was introduced so that the concentration in the groundwater was 0.02% or less, and the Rhodococcus jostii RHA1 strain was introduced into the groundwater with a bacterial count of 5.0 × 10 ⁸ cells / ml. The method according to [5] or [6], which is introduced so as described above.

According to the present invention, a volatile organochlorine compound can be efficiently decomposed only by using a bacterium having a predetermined ability and hydrogen peroxide.

The decomposition route of volatile organic chlorine compounds using oxygen is shown. The growth curves of the RHA1 strain in the presence of hydrogen peroxide of various concentrations are shown. The decomposition status of trichlorethylene by the RHA1 strain in the presence of hydrogen peroxide of various concentrations is shown. The decomposition status of trichlorethylene by the TDR12 strain (mutant strain of the RHA1 strain in which the degrading gene of the volatile organochlorine compound is eliminated) in the presence of various concentrations of hydrogen peroxide is shown. It shows the time course of hydrogen peroxide concentration under various conditions (control, catalase reagent, or RHA1 strain). The time course of the hydrogen peroxide concentration in the decomposition test of cis-1,2-dichloroethylene by the RHA1 strain at various concentrations in the presence of hydrogen peroxide at various concentrations is shown. The decomposition status of cis-1,2-dichloroethylene by the RHA1 strain at various concentrations in the presence of hydrogen peroxide at various concentrations is shown. The time course of the hydrogen peroxide concentration in the decomposition test of cis-1,2-dichloroethylene by the RHA1 strain at various concentrations in the presence of hydrogen peroxide is shown. The decomposition status of cis-1,2-dichloroethylene by the RHA1 strain at various concentrations in the presence of hydrogen peroxide is shown.

Hereinafter, the present invention will be described in detail.

The present invention is a method for purifying a volatile organic chlorine compound, which comprises a decomposition step of allowing a bacterium having a predetermined ability to act on the volatile organic chlorine compound in the presence of hydrogen peroxide to decompose the volatile organic chlorine compound. Regarding.

As the bacterium having a predetermined ability, a bacterium having a catalase-producing ability and an ability to decompose a volatile organic chlorine compound in the presence of oxygen is used. Preferably, a bacterium that is more resistant to hydrogen peroxide is used.

"Catalase" is an enzyme that breaks down hydrogen peroxide into oxygen and water.

A bacterium having "resistance to hydrogen peroxide" means a bacterium capable of producing catalase and converting hydrogen peroxide into oxygen even in the presence of hydrogen peroxide. Specifically, in the presence of 0.01% hydrogen peroxide, preferably in the presence of 0.05% hydrogen peroxide, more preferably in the presence of 0.1% hydrogen peroxide, even more preferably 0.5. It means a bacterium that can generate catalase and convert hydrogen peroxide into oxygen even in the presence of hydrogen peroxide at a concentration of%.

The oxygen used for decomposing the volatile organochlorine compound may be oxygen contained in the air or oxygen generated from hydrogen peroxide by the action of catalase.

In the method according to the present invention, the bacterium can efficiently decompose volatile organic chlorine compounds using oxygen (Fig. 1).

Bacteria of the genus Rhodococcus can be used as bacteria capable of producing catalase and decomposing volatile organochlorine compounds in the presence of oxygen. Bacteria of the genus Rhodococcus include Rhodococcus aichiensis, Rhodococcus aurantiacus, Rhodococcus baikonurensis, Rhodococcus baikonurensis, Rhodococcus bikonurensis, Rhodococcus bikonurensis, Rhodococcus bikonurensis, Rhodococcus bikonurensis (Rhodococcus chlorophenolicus), Rhodococcus chubuensis, Rhodococcus coprophilus, Rhodococcus corallinus, Rhodococcus corallinus, Rhodococcus corallinus, Rhodococcus corallinus, Rhodococcus corallinus, Rhodococcus corallinus, Rhodococcus corallinus Police (Rhodococcus erythropolis), Rhodococcus fascians, Rhodococcus globerulus, Rhodococcus jostii, Rhodococcus jostii, Rhodococcus koriences, Rhodococcus koredococcus Rhodococcus maanshanensis, Rhodococcus marinonascens, Rhodococcus maris, Rhodococcus opacus, Rhodococcus opacus, Rhodococcus rhodococcus (Rhodococcus rhodnii), Rhodococcus rhodochrous, Rhodococcus ruber, Rhodoc Rhodococcus terrae, Rhodococcus triatomae, Rhodococcus tukisamuensis, Rhodococcus wratislaviensis, Rhodococcus wratislaviensis, Rhodococcus wratislaviensis, Rhodococcus wratislaviensis ) Etc. can be mentioned.

Although not particularly limited, in order to decompose volatile organochlorine compounds more efficiently, it is preferable to use Rhodococcus jostii as a bacterium of the genus Rhodococcus, and Rhodococcus jostii is preferable. (Rhodococcus jostii) It is particularly preferable to use an RHA1 strain (hereinafter, simply referred to as "RHA1 strain"). The RHA1 strain can be obtained from the Product Evaluation Technology Infrastructure Organization (NBRC 108803).

The RHA1 strain is an aerobic bacterium isolated from a field contaminated with γ-hexachlorocyclohexane and is widely used for degrading polychlorinated biphenyls (PCBs) (eg, Patent No. 2990016). The RHA1 strain co-metabolizes PCBs with a biphenyl-degrading enzyme system. Biphenyls are biphenyldioxygenase (BphA), dihydrodiol dehydrogenase (BphB), 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), and 2-hydroxy-6-oxo-6-phenylhexa-2,4- It is converted to benzoic acid and 2-hydroxypenta-2,4-dienoic acid by a series of reactions consisting of dienoate hydrolase (BphD). 2-Hydroxypenta-2,4-dienoic acid produced in the reaction of BphD is converted to pyruvic acid and acetyl-CoA, and benzoic acid is converted to succinyl-CoA and acetyl-CoA via catechol and 3-oxyadipic acid. Once converted, they are eventually led to the citric acid cycle for use as a carbon and energy source.

The existence of multiple isozymes has been clarified for each of BphA, BphB, BphC, and BphD. In BphA, an isozyme composed of three types of dioxygenase components encoded by bphAaAb, etbAa1Ab1 and etbAa2Ab2 has been found. In BphB, two isozymes encoded by bphB1 and bphB2 have been found. In BphC, two isozymes encoded by bphC1 and etbC have been found. In BphD, three isozymes encoded by bphD, etbD1 and etbD2 have been found. These enzyme genes form five enzyme clusters and are distributed on the giant linear plasmid pRHL1 or pRHL2.

The RHA1 strain can decompose volatile organochlorine compounds by the hydroxylation reaction of biphenyldioxygenases (BphA and EtbA).

Examples of the volatile organochlorine compound include chlorinated ethylenes such as tetrachlorethylene, trichlorethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene and vinyl chloride monomer. Although not particularly limited, the method according to the present invention particularly targets trichlorethylene and cis-1,2-dichloroethylene.

When chlorinated ethylene (particularly trichlorethylene and cis-1,2-dichloroethylene) is decomposed using the RHA1 strain, vinyl chloride monomer is not produced in the decomposition process, so that secondary environmental pollution can be avoided. In addition, since the RHA1 strain can use the substance to be purified itself as an inducing substrate, it is not necessary to use harmful inducing substrates such as toluene and phenol, and further environmental pollution can be avoided. Furthermore, since the RHA1 strain does not need to use a substance that is difficult to dissolve in water such as methane as an inducing substrate, it can be purified by introducing only the RHA1 strain and hydrogen peroxide into an environment to be purified. ..

In the method according to the present invention, the bacterium decomposes volatile organic chlorine compounds using oxygen. Therefore, the method according to the present invention can be performed in an aerobic environment. The "aerobic environment" is an environment in which a sufficient amount of oxygen is present for the bacteria to decompose volatile organochlorine compounds. Examples of the aerobic environment include a ground environment in which oxygen is sufficiently present.

In the method according to the present invention, oxygen can be supplied from hydrogen peroxide by the action of catalase possessed by the bacterium. Therefore, the method according to the present invention can also be performed in a microaerobic environment with little oxygen or an anaerobic environment without oxygen. The "microaerobic environment" is an environment in which oxygen is present but not in a sufficient amount for the bacteria to decompose volatile organochlorine compounds. An "anaerobic environment" is an environment in which oxygen is completely absent. Examples of the microaerobic environment or the anaerobic environment include underground environments in which oxygen is not sufficiently present or not present at all, such as groundwater and underground soil.

For example, in one embodiment of the present invention, a method of injecting the bacteria and hydrogen peroxide into an underground environment contaminated with a volatile organochlorine compound to purify the underground environment is targeted. A further embodiment of the present invention covers a method of injecting the bacteria and hydrogen peroxide into groundwater containing a volatile organochlorine compound to purify the groundwater in situ.

Conventionally, when purifying pollution in the underground environment using aerobic bacteria, it has been necessary to introduce oxygen into the underground environment. Methods for introducing oxygen into the underground environment include injection of an oxygen sustained-release agent containing magnesium peroxide as the main component, injection of high-concentration oxygen water, injection of micro-nano-level fine oxygen bubbles, ozone injection, air sparging, etc. Are known. On the other hand, in the method according to the present invention, oxygen can be supplied from hydrogen peroxide by the action of catalase by injecting a bacterium capable of producing catalase and hydrogen peroxide. By using hydrogen peroxide, the efficiency of oxygen supply to the underground environment can be increased, and the cost of supplying oxygen to the underground environment can be reduced.

On the other hand, by injecting hydrogen peroxide into the underground environment, hydrogen peroxide not only produces oxygen by catalase generated by bacteria, but also hydrogen peroxide reacts with catalytic substances such as iron compounds in the ground (hydroxy radicals). Oxygen is also generated by the reaction). When a hydroxyl radical reaction occurs, hydroxyl radicals that are harmful to the bacterium are generated and the bacterium is killed. Since the present inventors have discovered that the catalase reaction by bacteria proceeds faster than the hydroxyl radical reaction in the ground, in order to convert hydrogen peroxide into oxygen by the catalase reaction as much as possible, a certain concentration of bacteria is mixed with hydrogen peroxide in the ground. The importance of introducing it to the above became clear.

Hydrogen peroxide is preferably injected into the underground environment so that sufficient oxygen is supplied to the bacteria to decompose the volatile organic chlorine compounds and the concentration of hydroxyl radicals is suppressed as much as possible. The specific hydrogen peroxide concentration is appropriately changed according to the type of bacteria used, the concentration of oxygen remaining in the groundwater, etc. However, if the hydrogen peroxide concentration is 0.02%, oxygen is sufficiently supplied. if present at a concentration of that time RHA1 strain groundwater 5 × 10 ⁸ cells / mL, can be reduced inhibition by hydroxy radicals RHA1 strain. Therefore, the hydrogen peroxide concentration in groundwater is 0.02% or less, and RHA1 strain it is preferable to adjust the injection amount to a concentration of more than 5 × 10 ⁸ cells / mL in groundwater. The lower limit of the hydrogen peroxide concentration in the groundwater may be, for example, 0.01%, 0.005%, 0.001% or the like. The upper limit of the RHA1 strain concentration in groundwater may be, for example, 1 × 10 ⁹ cells / mL, 5 × 10 ⁹ cells / mL, 1 × 10 ¹⁰ cells / mL, or the like.

Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited thereto.

[Example 1] Resistance evaluation test of RHA1 strain to hydrogen peroxide In this test, in order to evaluate the resistance of RHA1 strain to hydrogen peroxide, the RHA1 strain was placed in a succinic acid medium in which the hydrogen peroxide concentration was gradually changed. The growth status of hydrogen peroxide was confirmed.

The RHA1 strain was inoculated into 10 ml of LB liquid medium (1 g / l Bacto tryptone, 0.5 g / l Yeast extract, 0.5 g / l NaCl) and cultured overnight. The cells were collected from the culture medium by centrifugation (5,000 rpm, 5 minutes, 4 ° C), washed twice with 5 ml of W inorganic salt medium (Table 1), and then the cells were placed in 1 ml of W inorganic salt medium. Suspended. In a small L-shaped tube having a capacity of 5 ml, 5 ml of the culture solution was prepared with W inorganic salt medium so that the OD600 was 0.4. Succinic acid having a final concentration of 10 mM was added to the medium as a carbon source, and hydrogen peroxide was added so that the final concentrations were 0%, 0.01%, 0.05%, 0.1%, 0.5%, and 1.0%. A small L-shaped tube was placed in an automatic OD measurement incubator (ADVANTEC) and cultured at a rotation speed of 70 rpm and 30 ° C. The culture broth (absorbance at 660 nm) was automatically measured and recorded every hour.

The results of the test are shown in FIG. At a hydrogen peroxide concentration of 0%, a logarithmic growth phase was observed from 6 hours to 30 hours of culturing, and A660 reached about 1.3 in 40 hours, reaching a steady phase. Similar growth was observed at hydrogen peroxide concentrations of 0.01% and 0.05%. At a hydrogen peroxide concentration of 0.1%, the growth induction period was extended about three times and growth was delayed, but the growth rate during the logarithmic growth period was almost the same as when the hydrogen peroxide concentration was 0%. At a hydrogen peroxide concentration of 0.5%, a decrease in viability was observed, but the cells grew until A660 reached about 0.6 after 70 hours of culturing. At a hydrogen peroxide concentration of 1.0%, A660 decreased after 6 hours of culturing and did not grow as it was.

These results suggest that the RHA1 strain is resistant to hydrogen peroxide up to at least 0.5%. On the other hand, it has been reported that Escherichia coli is sensitive to hydrogen peroxide of 0.6 mM (≈0.002%). Therefore, it is judged that the resistance of the RHA1 strain to hydrogen peroxide is high.

[Example 2] Evaluation test of the effect of hydrogen peroxide on the trichlorethylene (TCE) resolution of the RHA1 strain In this test, in order to evaluate the effect of hydrogen peroxide on the trichlorethylene (TCE) resolution of the RHA1 strain, the hydrogen peroxide concentration was stepwise. A degradation test of trichlorethylene was carried out in a modified succinic acid medium.

200 ml of W inorganic salt medium containing 20 mM succinic acid was prepared, and RHA1 strain cultured in LB medium for 24 hours was added so that OD600 was 0.1, and cultured at 120 rpm and 30 ° C. for 24 hours. Then, 4 mmol (20 mM) of succinic acid was added to the culture broth, and the cells were further cultured for 24 hours. After culturing, the cells were collected by centrifugation (5,000 rpm, 5 minutes, 4 ° C.), and the cells were washed twice using 30 ml of W inorganic salt medium. In a 100 ml brown glass vial, 20 ml of a suspension having an OD600 of 10 (≈1.0 × 10 ^{8 cfu / ml) was prepared using W inorganic salt medium.} Trichlorethylene was added to the suspension to a final concentration of 10 mg / l, immediately sealed with a Teflon-coated butyl rubber stopper, and at 180 rpm and 30 ° C for 1 hour to stabilize the vapor-liquid equilibrium of trichlorethylene. It was shaken. During the test period, the vials were stored in an incubator and continued to shake at 180 rpm and 30 ° C. Immediately after the measurement on the 0th day, hydrogen peroxide was added by injecting directly into the vial from a rubber stopper so that the final concentration was 0%, 0.01%, 0.05%, 0.1%, 0.5% with a microsyringe. Trichloroethylene was quantified by taking a 400 μl gas phase sample from the top of the vial using a gas tight syringe and analyzing it with a gas chromatography-flame ionization detector (GC-FID). In the analysis, the area was calculated from the peak derived from trichlorethylene, and the proportion of trichlorethylene remaining in the vial was determined for each number of days based on the peak area at the start of measurement.

The results of the test are shown in FIG. The RHA1 strain decomposed trichlorethylene up to a hydrogen peroxide concentration of 0.1% in the same manner as in the absence of hydrogen peroxide. Although a slight decrease in trichlorethylene resolution was observed at a hydrogen peroxide concentration of 0.5%, trichlorethylene was decomposed to about 95% in 3 days.

As a comparative control test, a trichlorethylene degradation test was conducted in the presence of hydrogen peroxide using a bphT1 and bphT2 double gene disruption strain (TDR12 strain) derived from the RHA1 strain. The expression of biphenyldioxygenases BphA and EtbA, which are responsible for the trichlorethylene degradation of the RHA1 strain, is positively regulated at the transcriptional level by two sets of BphS-BphT two-component control systems. The TDR12 strain derived from the RHA1 strain whose trichlorethylene resolution was blocked by the bphT1 and bphT2 double gene disruption was used in this study.

As a result of conducting the test under the same conditions as above, no decomposition of trichlorethylene was observed up to a hydrogen peroxide concentration of 0.1% (Fig. 4). Decomposition of about 40% of trichlorethylene was observed at a hydrogen peroxide concentration of 0.5%. From the above results, it was shown that the TDR12 strain does not decompose trichlorethylene. The partial decomposition of trichlorethylene at a hydrogen peroxide concentration of 0.5% was presumed to be the chemical decomposition of trichlorethylene (hydroxy radical reaction) by hydrogen peroxide catalyzed by cells.

From the above results, in a medium containing hydrogen peroxide (an environment in which no catalytic substance is present such as soil), if the hydrogen peroxide concentration is 0.1% or less, the RHA1 strain is not inhibited by hydrogen peroxide. Was shown to degrade trichlorethylene.

[Example 3] Confirmation test of catalase-producing ability of RHA1 strain In this test, RHA1 strain and commercially available catalase were used to confirm whether hydrogen peroxide was decomposed by catalase produced from RHA1 strain in actual contaminated groundwater. The test was performed using reagents.

The RHA1 strain was inoculated into 100 ml of LB liquid medium (1 g / l Bacto tryptone, 0.5 g / l Yeast extract, 0.5 g / l NaCl) and cultured overnight. Prepare 200 ml of W inorganic salt medium (SYW medium) supplemented with 20 mM succinic acid, 1 g / l yeast extract, and 5 mM ammonium sulfate, and add the RHA1 strain cultured overnight in LB liquid medium to 100. The cells were cultured at rpm and 30 ° C. for 48 hours. The cells after culturing were collected by centrifugation (5,000 rpm, 10 minutes, 4 ° C.), and the cells were washed with W inorganic salt medium and used for the decomposition test.

Table 2 shows the test conditions. Condition 2 was a catalase solution derived from bovine liver, and condition 3 was a RHA1 strain. Catalase was dissolved in phosphate buffer and the amount of catalase solution required for the complete decomposition of 0.02% hydrogen peroxide was added. Condition 1 was a control group to which the RHA1 strain and the catalase reagent were not added. After adding groundwater and bacterial solution to the vials to be used, hydrogen peroxide and catalase reagents were added to the vials. After sealing with a butyl rubber stopper and an aluminum seal, the mixture was manually stirred and homogenized, and then statically cultured in a constant temperature bath at 20 ° C. Multiple vials under each condition were prepared, and after 0, 3, and 7 days had passed, the vials were opened and the supernatant was collected with a pipette for analysis.

FIG. 5 shows the change over time in the hydrogen peroxide concentration under each condition. Under condition 3 to which the RHA1 strain was added and condition 2 to which the catalase solution was added, the total amount of hydrogen peroxide was consumed immediately after the start of the test. From this result, it was shown that the RHA1 strain produced catalase and decomposed hydrogen peroxide. On the other hand, hydrogen peroxide remained under condition 1 in which the RHA1 strain and the catalase solution were not added.

From the above results, it was shown that the RHA1 strain produced catalase and instantly decomposed hydrogen peroxide to produce oxygen.

[Example 4] Sis-1,2-dichloroethylene decomposition test by simultaneous introduction of RHA1 strain and hydrogen peroxide using actual contaminated groundwater In this test, RHA1 strain and hydrogen peroxide were introduced simultaneously in the groundwater of the actual contaminated site. Then, it was examined whether cis-1,2-dichloroethylene (cis-DCE) was decomposed by the oxygen supply accompanying the decomposition of hydrogen peroxide.

Table 3 shows the test conditions. After adding a small amount of soil (sandy soil), groundwater, and the bacterial solution prepared in the same manner as in Example 3 into the vials to be used, the vial is filled with cis-DCE concentrate and hydrogen peroxide. As described above, the input amount was calculated in advance and added. After sealing with a butyl rubber stopper and an aluminum seal, the soil and groundwater were manually stirred and homogenized, and statically cultured in a constant temperature bath at 20 ° C. Multiple vials under each condition were prepared, and after 0, 1, 3, and 7 days had passed, the vials were opened and the supernatant was collected with a pipette for analysis. The amount of cells was evaluated under three conditions of ^{1.0 × 10 8} cells / ml and 5.0 × 10 ⁸ cells / ml without adding RHA1 strain. In addition, the final concentration of hydrogen peroxide to be added in the liquid phase was 0.02% and 0.1%.

FIG. 6 shows the change over time in the hydrogen peroxide concentration under each condition. As a result, the condition that hydrogen peroxide is completely consumed by the catalase reaction immediately after the start of the test (that is, the condition that the hydroxyl radical reaction does not occur due to the residual hydrogen peroxide and the RHA1 strain is not inhibited) is condition 1. It was confirmed that -2, condition 1-3, and condition 1-6. Under conditions 1-5, the amount of RHA1 strain introduced was small relative to the hydrogen peroxide concentration, and it is considered that the RHA1 strain was damaged by the hydroxyl radical reaction and catalase was no longer produced.

FIG. 7 shows the residual rate of cis-DCE under the condition that hydrogen peroxide was completely consumed by the catalase reaction immediately after the start of the test. Under condition 1-2, the hydrogen peroxide introduced immediately after the start of the test was converted to oxygen, but the decomposition of cis-DCE was hardly observed, so it was considered that the concentration of the introduced RHA1 strain cells was insufficient. Comparing conditions 1-3 and 1-6, which have similar RHA1 strain cell concentrations, it was shown that cis-DCE decomposed better at 0.02%, which has a lower hydrogen peroxide concentration, and in water. It was shown that if the hydrogen peroxide concentration is 0.02%, sufficient oxygen required for decomposition can be secured.

Based on the above results, the condition for converting the entire amount of hydrogen peroxide into oxygen in a short time by the catalase reaction (that is, the condition that the introduced RHA1 strain is not easily inhibited by the hydroxyl radical reaction) is that the hydrogen peroxide concentration is the required amount of oxygen. It is important to reduce the amount of radicals as much as possible within the range that can be secured, and in this test, it is optimal to set the hydrogen peroxide concentration to 0.02% for the ^{number of RHA1 strains of 5 × 10 8 cells / mL.} Was shown.

[Example 5] Sis-1,2-dichloroethylene decomposition test by adding RHA1 strain and hydrogen peroxide in an environment simulating an actual contaminated aquifer In this test, an aquifer (soil and groundwater) at an actual contaminated site is present. In a simulated environment), RHA1 strain and hydrogen peroxide were added, and it was examined whether cis-DCE was decomposed.

Table 4 shows the test conditions. Soil, groundwater (set so that the gas phase part is almost eliminated under all conditions), and the bacterial solution obtained by culturing and washing the RHA1 strain in the same manner as in Example 3 are added to the vial, and then the cis-DCE concentrate and hydrogen peroxide are added. Was added, and the mixture was sealed with a butyl rubber stopper. In this test, the amount of soil was changed from 1 g to 50 g as compared with the test of Example 4. The sealed vial was manually agitated and homogenized with soil and groundwater, and then statically cultured in a constant temperature bath at 20 ° C. Multiple vials were prepared for each condition, and after 0, 3, and 7 days, the supernatant was collected and analyzed in a state where the bottle was opened and allowed to stand. In this test, 0.02% hydrogen peroxide was added at a final concentration under the condition that about 40% of the bottle was immersed in soil, and the amount of cis-DCE decomposed by the amount of RHA1 strain introduced was evaluated.

FIG. 8 shows the change over time in the hydrogen peroxide concentration under each condition, and FIG. 9 shows the residual rate of cis-DCE. From FIG. 8, the difference in the hydrogen peroxide concentration immediately after the start under each condition is that the hydrogen peroxide was consumed by the reaction with the catalyst substance in the soil and the reaction with the catalase produced by the RHA1 strain at the moment when the hydrogen peroxide was added. , There was a difference in the initial concentration. Comparing the condition 1-1 of Example 4 with the condition 2-1 of this test in which 0.02% of hydrogen peroxide was added at the final concentration and the RHA1 strain was not introduced, the condition 2-1 with a large amount of soil added was 3 Hydrogen peroxide disappeared in days. It was shown that the consumption of hydrogen peroxide by the hydroxyl radical reaction became faster due to the increase in the amount of soil and the increase in the catalyst substance. Under condition 2-2, cis-DCE did not decrease after the 3rd day when hydrogen peroxide disappeared, but under condition 2-3, it was clearly cis compared with condition 2-1 in which the RHA1 strain was not introduced. -Since a decrease in DCE was confirmed, it was inferred that biodegradation was performed by the RHA1 strain. Hydrogen peroxide was decomposed into oxygen within 3 days, but biodegradation by the RHA1 strain after the 3rd day seems to have proceeded by consuming the oxygen remaining in the vial.

^{Based on the above results, 5 × 10 8} cells / mL or more of RHA1 strain was introduced into the aquifer with respect to the final concentration of 0.02% hydrogen peroxide addition, and the hydrogen peroxide in the groundwater was completely eliminated within 3 days. It was shown that cis-DCE can be purified by introducing the RHA1 strain.

Claims (1)

Bacteria having the ability to produce catalase and the ability to decompose volatile organochlorine compounds in the presence of oxygen are allowed to act on the volatile organochlorine compounds in the presence of hydrogen peroxide to decompose the volatile organochlorine compounds. Including the decomposition step, the bacterium is a Rhodococcus jostii RHA1 strain, which is a bacterium belonging to the genus Rhodococcus.
The volatile organic chlorine compound is contained in the groundwater, and the groundwater is purified in place to purify it.
The hydrogen peroxide is introduced so that the concentration in the groundwater is 0.02% or less, and the Rhodococcus jostii RHA1 strain is introduced into the groundwater so that the number of bacteria in the groundwater is 5.0 × 108 cells / ml or more. A method of purifying a primary organochlorine compound, which is introduced in the above manner.
To ask.

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